1. Increased movement of GPR55 KO cells in association with epithelial cells.
Increased movement of GPR55 KO cells in association with epithelial cells. (A) Representative z-projection view of small intestinal villi of GPR55 Het (top) and KO (bottom) mice, showing CD8α-PE–labeled cells (red) and cell nuclei (Hoechst, blue). Lines show tracks of labeled IELs. Images are representative from more than six mice in each genotype. Scale bars, 20 μm. (B) Representative z-projection view of small intestinal villi of TCRδ-eGFP+ GPR55 Het (top) and KO (bottom) mice, showing eGFP (green) and cell nuclei (Hoechst, blue). Lines show tracks of GFP+ cells. Circles and ovals in (A) and (B) show regions of tracks that are localized in close association with epithelial cells. Images are representative from more than seven mice in each genotype. Scale bars, 40 μm. (C) Representative snapshots from single z-plane movies. Left two panels from GPR55 Het and right two panels from GPR55 KO. Close association movement along epithelium observed as overlap of eGFP signal with the fluorescence signal of the epithelial cell nuclei labeled with Hoechst (circles in top right). Scale bars, 10 μm. (D and E) Representative multiple z-stack projection view (D) and single z-plane view (E) of small intestinal villi of mixed BM chimeras of the type described in Fig. 4D. Red cells are Het (red tracks), whereas red and green double-positive cells are KO (green tracks). Ovals in (D) (yellow in Het and orange in KO) show regions of tracks that are localized in close association with epithelial cells. Dashed white lines in (E) indicate the boundaries of the Hoechst-labeled epithelial cell nuclei (ECN) and the PMS used for the quantitation. Images are representative from six mice. Scale bars, 20 μm. (F) Speed of GPR55 Het and KO γδT IELs in mixed BM chimeras. (G and H) Percentage of time in close association with epithelial cells and time of each association (G), frequency of lateral intercellular space (LIS) probing, and time of each probing (H) of GPR55 Het and KO cells in mixed BM chimeras. Combined data from six movies from six different chimeric mice. (I) Induction of Pkr and Usp18 mRNA in the small intestine of GPR55 Het and KO mice treated with anti-CD3 antibody (Ab) for 3 hours, plotted relative to Hprt. HPRT, hypoxanthine-guanine phosphoribosyltransferase. Combined data from two independent experiments (n = 9 in each group). ***P < 0.001, **P < 0.01, *P < 0.05, n.s. P > 0.05 by Student’s t test.
Hayakazu Sumida et al. Sci. Immunol. 2017;2:eaao1135
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